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Basic Research
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Laminin β2 Gene Missense Mutation Produces Endoplasmic Reticulum Stress in Podocytes

Ying Maggie Chen, Yuefang Zhou, Gloriosa Go, Joseph T. Marmerstein, Yamato Kikkawa and Jeffrey H. Miner
JASN August 2013, 24 (8) 1223-1233; DOI: https://doi.org/10.1681/ASN.2012121149
Ying Maggie Chen
*Renal Division and
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Yuefang Zhou
*Renal Division and
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Gloriosa Go
*Renal Division and
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Joseph T. Marmerstein
*Renal Division and
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Yamato Kikkawa
†Laboratory of Clinical Biochemistry, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan
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Jeffrey H. Miner
*Renal Division and
‡Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri; and
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    Figure 1.

    There are three different levels of NEPH-C321R-LAMB2 transgene expression in the three independent lines. These are revealed by (A and B) quantitative confocal immunofluorescence and (C) in situ hybridization. Transgenes were on the Lamb2−/− background in all cases. (A) Quantitative confocal immunofluorescence showed different levels of transgene-derived rat C321R-LAMB2 and R246Q-LAMB2 proteins in the GBM. LAMB2 protein accumulation in the GBM was detected at ∼3 weeks of age by colocalization of mutant LAMB2 (green) and agrin (red) in overlay images (merge). As shown in the histogram, significant differences in total fluorescence intensity were observed in glomeruli of the different groups (n=20–25 glomeruli for each line). **P<0.001, *P<0.05 by t test. Scale bar, 10 µm. (B) Panels from our previous paper19 show that Tg-WT mice exhibited much higher rat β2 in the GBM compared with R246Q-TgLo mutants. To avoid image saturation and the resulting quantification errors, different laser intensities and gains were used for the upper and lower panels; R246Q-TgHi staining was used as a common reference for comparison between R246Q-TgLo mutants and the Tg-WT mice. Original magnification, ×800. (C) Transgene-derived mRNA was easily detected by in situ hybridization in C321R-TgMed, C321R-TgHi, and Tg-WT mice but not C321R-TgLo or R246Q-TgLo mutants (at approximately 3 weeks). Scale bars, 100 µm.

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    Figure 2.

    Proteinuria in Lamb2−/−; NEPH-C321R-LAMB2 mutant mice correlates inversely with the level of transgene expression before the first month of age. Urinary protein/creatinine ratios (gram/gram) between 3 and 4 weeks of age in the three lines of Lamb2−/−; C321R-LAMB2 mice and their WT and nontransgenic Lamb2−/− littermates are shown in the graph as mean ± SD. **P<0.001. NS, not significant. Statistical analysis was done by ANOVA with the Tukey multiple comparison test.

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    Figure 3.

    Ectopic deposition of laminins α1, α2, and β1 in the GBM is associated with reduced expression of β2 in the GBM of Lamb2−/−; C321R-LAMB2 mice. Frozen kidney sections from TgHi mutants, their WT and nontransgenic Lamb2−/− littermates, and age-matched Lamb2−/−; Tg-WT mice were stained for laminin chains at 3 weeks. (A and E) The normally mesangial laminins α1 and α2 were ectopically deposited in the (B and F, arrows) TgHi and (C and G, arrows) Lamb2−/− GBM but not (D and H) Tg-WT GBM. I–L show merged images for which overlapping laminin β1 (red) and nidogen (green) signals appear (J and K, arrows) yellow in the GBM of Lamb2−/−; TgHi and Lamb2−/− mice as well as (I–L) the mesangium of all mice, where both proteins are normally present. Additional data are shown in Supplemental Figure 1. Scale bar, 20 µm.

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    Figure 4.

    Light and electron microscopy analyses of kidneys from the three lines of Lamb2−/−; NEPH-C321R-LAMB2 mice and their Lamb2−/− and WT littermates at 3 weeks show that glomerulosclerosis and mild GBM thickening in Lamb2−/− mice are attenuated by increased expression of C321R-LAMB2 in the GBM. (A) Paraffin sections of WT and mutant kidneys were stained with H&E (HE) and PAS. Lamb2−/− mice showed (d) glomerulosclerosis, (h, arrow) prominent renal tubular protein casts, and (h) loss of brush borders compared with (a and e) the WT littermates. TgLo Lamb2−/−; C321R-LAMB2 mice exhibited (b) MM expansion, (f, arrow) fewer protein casts, and (f) partial loss of brush borders. (c and g) No obvious pathology was detected in TgHi mutants. Scale bars, 20 µm in a–d; 100 µm in e–h. (B) TEM showed diffuse FP effacement (*) and mild GBM thickening (arrow) in (e) Lamb2−/− mice compared with (a) controls. FP effacement (*) was observed in (b–d) all mutant lines, with some GBM outpocketing in the (b, arrows) TgLo and (c, arrows) TgMed mutants. Insets in b and c magnify regions between the two arrows. (f–h) Focal areas with intact foot processes were also found in the three mutant Tg lines. Scale bar, 500 nm.

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    Figure 5.

    Increased level of C321R-LAMB2 in the GBM is associated with less interstitial fibrosis at 6 weeks. TEM analysis showed irregular GBM thickening (arrows) and diffuse FP effacement (*) in TgLo, TgMed, and TgHi Lamb2−/−; C321R-LAMB2 mice. Scale bar, 500 nm. Gomori’s Trichrome staining revealed less interstitial fibrosis in TgMed and TgHi mutants compared with TgLo mutants (arrows). Scale bar, 100 µm.

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    Figure 6.

    Secretion of the mutant laminin β2 fragment/Gluc fusion protein is inhibited and podocyte ER stress is induced in TgHi Lamb2−/−; C321R-LAMB2 mice. (A, arrow) The WT but not the C321R-LAMB2/Gluc fusion protein was secreted from stably transfected HEK293T cells. (B) This finding was reflected by luciferase activity in the media (**P<0.001 by t test). The data are presented as mean ± SD of three independent media samples. (C) The C321R/Gluc fusion protein was retained intracellularly compared with the WT fusion protein. (D) Quantitative RT-PCR did not show a significant difference in Lamb2 mRNA levels between the WT and C321R cell clones. Data are presented as mean ± SD of fold changes of four independent samples from each clone. P>0.05 by t test. (E) Colocalization of the (a) WT/Gluc or (b) C321R LAMB2/Gluc fusion protein with the (c and d) ER marker PDIA3 in the 293T-Gluc cells using confocal microscopy. (e and f) Nuclei were counterstained with Hoechst 33342 (blue). (a, c, and e) The WT fusion protein seemed to be transiently associated with PDIA3, but (b, d, and f) the mutant was completely colocalized with PDIA3, indicating accumulation in the ER. (c and d) The mutant protein also induced expression of PDIA3. Scale bar, 10 µm. (F) Cell lysates of 293T-Gluc clones were subjected to Western blot analysis. The C321R but not the WT fusion protein increased BiP expression (arrow). Tunicamycin (TM) -treated cells served as positive controls. (G) Frozen kidney sections from mice of the indicated genotypes were examined by dual immunofluorescence staining of BiP and WT-1 at 3 weeks. Compared with the very low level expression of (a, e, and i) BiP in the control podocytes, BiP upregulation was detected in podocytes of (c, g, and k) TgHi glomeruli and (d, h, and l) some Lamb2−/− glomeruli but not (b, f, and j) podocytes of Tg-WT glomeruli. Scale bar, 20 µm.

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    Figure 7.

    C321R-LAMB2 causes podocyte ER distention, upregulation of CHOP, and activation of desmin in podocytes at 3 weeks. (A) TEM showed that rER in the podocytes of (b, arrows) TgHi Lamb2−/−; C321R-LAMB2 and (c, arrows) Lamb2−/− mice was dilated compared with rER in the (a) podocytes of age-matched Lamb2−/−; Tg-WT controls. The swollen, ribosome-studded (b, arrows) tubular structures or (c, arrows) vesicles identify the rER. Scale bar, 500 nm. (B) Double immunofluorescence staining for (a–c) CHOP and (d–f) WT-1 on frozen kidney sections revealed podocyte induction of CHOP in (b, e, and h) Lamb2−/−; TgHi and (c, f, and i) Lamb2−/− glomeruli but not (a, d, and g) Tg-WT glomeruli. Scale bar, 20 µm. (C) Frozen sections of Lamb2−/−; Tg-WT, Lamb2−/−; TgHi, and Lamb2−/− kidneys were stained for (a–c) podocin and (d–f) desmin and (g–i) merged. Compared with (a, d, and g) controls, desmin expression was activated in the podocytes of (b, e, and h, arrows) mildly proteinuric TgHi mutants and (c, f, and i, arrows) highly proteinuric Lamb2−/− mutants. Scale bar, 20 µm.

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    Figure 8.

    TUDCA treatment increases secretion of the C321R-LAMB2/Gluc fusion protein in vitro. Secretion of (A) WT or (B) C321R mutant β2 fragment/Gluc fusion proteins from stably transfected 293T cells was measured by luciferase assays at different time points. The data are presented as mean ± SD of three independent media samples at 24, 48, or 72 hours. (A) TUDCA did not affect secretion of the WT fusion protein (P>0.05 by t test). (B) Secretion of the C321R fusion protein was enhanced by the addition of 1 mM TUDCA from 24 to 72 hours (**P<0.001 by t test). Note that the medium assayed in A was diluted 1:20.

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Journal of the American Society of Nephrology: 24 (8)
Journal of the American Society of Nephrology
Vol. 24, Issue 8
August 2013
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Laminin β2 Gene Missense Mutation Produces Endoplasmic Reticulum Stress in Podocytes
Ying Maggie Chen, Yuefang Zhou, Gloriosa Go, Joseph T. Marmerstein, Yamato Kikkawa, Jeffrey H. Miner
JASN Aug 2013, 24 (8) 1223-1233; DOI: 10.1681/ASN.2012121149

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Laminin β2 Gene Missense Mutation Produces Endoplasmic Reticulum Stress in Podocytes
Ying Maggie Chen, Yuefang Zhou, Gloriosa Go, Joseph T. Marmerstein, Yamato Kikkawa, Jeffrey H. Miner
JASN Aug 2013, 24 (8) 1223-1233; DOI: 10.1681/ASN.2012121149
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